If one wants to examine the effect of reagents, active substances or other influences on an organism, these experiments are often carried out in cell culture in in vitro models. The various enzymatic activities of cells yield information on their condition and physiological properties.
Often attempts are made to detect the activation of genes by means of so-called reporter gene tests. In these tests the gene for a certain detection enzyme (=reporter gene) is placed behind a promoter whose activation one wishes to detect. Secreted enzymes such as for example the secretory alkaline phosphatase, the activity of which is then tested with the enzyme substrate 4-nitrophenyl phosphate, can also be used for this as reporter genes. This substrate is converted by this enzyme into the product 4-nitrophenol which absorbs light at a wavelength of 405 nm.
If one wishes to genetically modify cells, one has to firstly transport the appropriate plasmid DNA through the plasma membrane and then into the cell nucleus with the aid of suitable methods. The methods used for this such as lipofection must be optimized in each case for the relevant cell line. Plasmids containing reporter genes such as the secretory alkaline phosphatase are also frequently used in these cases in which the aim is to optimize the transfectability of different cells. The success of the transfection can then be checked on the basis of the measured enzyme activity.
Different methods are also very frequently used to measure the cytotoxic properties of a substance. A number of tests for determining cytotoxicity may be found in the literature.
Many of these tests are based on the property of cytotoxic substances to damage the cell membrane. Enzymes which can be detected in the cell culture supernatant by an enzymatic test are released from cells whose plasma membrane has been damaged. The amount of released enzymes is proportional to the number of damaged cells. Such enzyme release tests have been described for glutamate-oxaloacetate transaminase, for glutamate pyruvate transaminase, for arginosuccinate lyase and for alkaline and acid phosphatase (Masanet, J., Gomez-Lechon, M. J., and Castell, J. V., Toxic. in Vitro 2 (1988) 275-282; Martin, Angela and Clynes, Martin, In Vitro Cell Dev. Biol. 27A (1991) 183-184). The release of alkaline phosphatase from human embryonic fibroblasts caused by lymphocytes was for example tested in this manner (Szekeres, Julia, Pacsa, A. S, and Pejtsik, B., J. Immun. Meth. 40 (1981) 151-154). However, lactate dehydrogenase is used most frequently for enzyme release tests since the aforementioned enzymes are often difficult to determine due to their small amount in many cells. In contrast to other enzymes lactate dehydrogenase is a very stable cytoplasmic enzyme which is present in all cells. It is very rapidly released from cells with a damaged plasma membrane and can be easily detected in the culture supernatant (Decker, Thomas and Lohmann-Matthes, Marie-Luise, J. Immun. Meth. 15 (1988) 61-69; Korzeniewski, Carol and Callewaert, Denis M., J. Immun. Meth. 64 (1983) 313-320).
In this test NAD+ is reduced in a first step to NADH/H+ by the LDH-catalysed conversion of lactate to pyruvate. In the second step a second enzyme (=catalyst diaphorase) transfers the H/H+ from NADH/H+ to the tetrazolium salt INT (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride) which is reduced in this process to the formazan (FIG. 1). The formazan dye is water-soluble and has a broad absorption maximum at 500 nm, while the substrate INT does not absorb at this wavelength (FIG. 2).
In other methods for measuring cytotoxicity fluorescent substrates such as resazurin are used instead of colorimetric enzyme substrates to measure LDH (Cytotox-One™ assay from the Promega Company, Madison, Wis.) or glucose-6-phosphate dehydrogenase (Vybrant Assay from the Molecular Probes Company from Eugene, Oreg.) in a similar reaction mixture. In other methods released glyceraldehyde-3-phosphate dehydrogenase is used to synthesize ATP by a coupled enzyme reaction. The ATP that is formed in this reaction is used in a bioluminescence reaction with luciferin and luciferase in which a measurable light signal is generated (Corey, Michael J., et al., J. Immun. Meth. 207 (1997) 43-51).
In addition to the tests for measuring a destroyed plasma membrane, the physiological activity and proliferation rate of cells is often also determined by means of their ability to reduce certain colour substrates. Colour substrates are used for this which change their colour when they are reduced in such a manner that only the product absorbs light of a certain wavelength which is then measured. Known reagents for this are for example tetrazolium salts such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (sodium 3′-[(1-phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)-benzylsulfonic acid hydrate) and WST-1 which are offered by Roche Applied Science (Mannheim, Germany) under the names Cell Proliferation Kit I (MTT), Cell Proliferation Kit II (XTT) and Cell Proliferation Reagent WST-1.
In addition to the tetrazolium salts, the blue dye resazurin is also used which is converted on reduction into the red strongly fluorescent dye resorufin which can be measured calorimetrically as well as fluorimetrically (Lancaster et al., U.S. Pat. No. 5,501,959).
WO 2003/089635 describes a combination of reagents for determining released LDH with the substrates resazurin or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, internal salt). The reduced form of MTS is measured colorimetrically and the reduced form of resazurin is measured colorimetrically or preferably fluorimetrically. In the case of this combination of reagents it is also suggested that a stop reagent should be added which stops the reaction. A soap or a detergent or a strong base such as NaOH is suggested as the stop reagent. A 3% SDS solution is recommended as the soap or detergent. A person skilled in the art is very familiar with the protein-denaturing properties of SDS where the reaction is stopped due to the denaturation of LDH. NaOH stops the reaction by changing the pH. However, only a very few known detergents or soaps result in a reaction stop as shown by the examples given below. The teaching in WO 2003/089635 would thus be to use such detergents or soaps which would result in a reaction stop i.e. which have protein-denaturing properties. Furthermore, with regard to the soaps or detergents stated in this patent no indication is given that they can be used to reduce light scattering or absorption caused by cells and thus to increase the sensitivity of calorimetric measurements.
In all methods in which an enzyme activity is detected calorimetrically in the presence of cells, the problem arises especially in the case of high cell counts that the light is scattered and absorbed by the cells in the entire wavelength range of visible light. Thus for example in the method described by Decker and Lohmann-Matthes for measuring released lactate dehydrogenase it is pointed out that in the case of high cell counts it is better to transfer the cell-free supernatants from the cell culture plate into new reaction vessels because of the observed high absorption of the cells and to measure the enzymatic activity in these reaction vessels. This procedure requires a centrifugation of the culture plates and a careful removal of the culture supernatant. These are additional working steps which make it more difficult to use the method for high throughput analysis for example with the aid of appropriate robots. In addition the cells may be damaged by the centrifugation or the removal of the supernatant thus releasing cytoplasmic enzymes which can interfere with the test result.
In the other methods in which a fluorimetric or a chemiluminescent measurement is carried out it is not possible to work with the widely used colorimetric measurement instruments especially for standard microtitre plates, the so-called ELISA readers, which considerably restricts the use of these substrates. In the case of chemiluminescent substrates the signal is often stable for only a relatively short period which requires a rapid measurement directly after the end of the test.
In the method published in WO 2003/089635 for determining released LDH using the substrates resozurin or MTS, these substrates are also described for colorimetric measurements. NaOH or SDS is claimed as a stop reagent. However, it must be noted that the colorimetric measurement of reduced resazurin only allows a low sensitivity. The use of NaOH or SDS with tetrazolium salts such as MTS is disadvantageous since NaOH leads to a precipitation of reduced tetrazolium salts (see example 2 of this application). Also SDS is not preferred since in this case tetrazolium salts are surprisingly bleached out relatively rapidly (see example 2 of this application).